Eects of the inhibition of p38/RK MAP kinase on induction of ®ve fos and jun genes by diverse stimuli Catherine A Hazzalin 1 , Ana Cuenda 2 , Eva Cano 1 , Philip Cohen 2 and Louis C Mahadevan 1 1 Nuclear Signalling Laboratory, Developmental Biology Research Centre, The Randall Institute, King's College London, 26-29 Drury Lane, London WC2B 5RL; 2 MRC Protein Phosphorylation Unit, Department of Biochemistry, Medical Sciences Institute, University of Dundee, DD1 4HN, UK The ERK, JNK/SAPK and p38/RK MAP kinase subtypes are dierentially activated by physiological, pharmacological and stress stimuli; all three subtypes are implicated in immediate-early (IE) gene induction by these agents. Here, we have asked whether inhibition of a single MAP kinase subtype under these conditions would generally alter induction of several IE genes in a similar way or whether this would dierentially up- and down-regulate particular IE genes, an issue which bears on the question of whether individual MAP kinases are strictly targeted to speci®c IE genes, or whether they might catalyse phosphorylation events that aect several IE genes in the same way. SB 203580, an inhibitor of p38/RK, has been used to analyse the role of this kinase in the induction of ®ve IE genes (c-fos, fosB, c-jun, junB and junD) under diverse conditions of stimulation. In C3H 10T cells, p38/RK and its downstream kinase MAPKAP K-2 are activated by all stimuli used with the exception of TPA. The speci®city of SB 203580 as a p38/RK inhibitor in these cells is demonstrated; it does not aect ERKs or JNK/SAPKs but does result in a small increase in the activity of the upstream kinase MKK6, the principal p38/RK activator in these cells. We ®nd that inhibition of p38/RK under these conditions produces general eects on all ®ve IE genes as a group in three ways. First, induction of all ®ve genes in response to okadaic acid or tumour necrosis factor-a (TNF-a) is not signi®cantly altered by SB 203580. Second, in cells stimulated with anisomycin or U.V. radiation, SB 203580 potently inhibits all of the induced IE genes. Finally, SB 203580 enhances induction of all ®ve IE genes in EGF-treated cells; these enhanced mRNA levels are not due to stabilisation of labile mRNA transcripts. The signi®cance of these results to current thinking on the relationship between distinct MAP kinase subtypes and speci®c IE genes is discussed. Keywords: MAP kinases; p38/RK; fos, jun; SB 203580 Introduction Three broad classes of stimuli elicit signalling responses and immediate-early (IE) gene transcription in diverse cell types. These are (i) physiological agents such as growth factors (reviewed in Marshall, 1995) and cytokines (Freshney et al., 1994; Sluss et al., 1994); (ii) stresses such as U.V. irradiation (Hibi et al., 1993; Kyriakis et al., 1994; Gupta et al., 1995; Livingstone et al., 1995; van Dam et al., 1995), chemical and heat shock (Rouse et al., 1994), and (iii) pharmacological compounds such as TPA (Cano et al., 1995), okadaic acid (Cano et al., 1995; reviewed in Cohen et al., 1990) and anisomycin (Edwards and Mahadevan, 1992; Cano et al., 1995; Hazzalin et al., 1996). Although there is considerable overlap in the signalling and gene induction responses elicited by these agents, the precise quantitative response of MAP kinase subtypes and individual IE genes appears characteristic for each agent (Cano et al., 1995; reviewed in Cooper, 1994; Cano and Mahadevan, 1995; Seger and Krebs, 1995). The only known signalling responses activated in common by all three classes of stimuli are the activation of p70/85 S6k and phosphorylation of S6, the dierential activation of MAP kinase subtypes and the nucleosomal response i.e. the rapid phosphorylation of histone H3 (Mahadevan et al., 1991; Barratt et al., 1994a) and HMG-14 (Barratt et al., 1994b) on serine residues in their NH-termini (Cano et al., 1995). However, p70/85 S6k activation can be ablated with rapamycin with no consequence to nucleosomal or IE gene responses (Kardalinou et al., 1994), leaving the MAP kinases as the only signalling cascade currently indissociable from nucleosomal and gene induction events. The prototypical MAP kinases ERK-1 and ERK-2 are de®ned by the motif TEY in the activation domain, within which threonine and tyrosine are phosphory- lated by an upstream kinase MEK for activation (reviewed in Cooper, 1994; Cano and Mahadevan, 1995; Seger and Krebs, 1995). The second subtype JNK/SAPKs, are characterised by the sequence TPY in the activation domain (DeÂrijard et al., 1994; Kallunki et al., 1994; Kyriakis et al., 1994), while the third, p38/ RK (p38, Han et al., 1994; CSBP, Lee et al., 1994; RK, Rouse et al., 1994; p40, Freshney et al., 1994; Mxi2, Zervos et al., 1995, referred to hereafter as p38/RK), has the motif TGY in this position. More recently, other variants of p38/RK, called SAPK3 (Mertens et al., 1996) and p38b (Jiang et al., 1996) have been identi®ed. These subtypes may be activated in parallel but to distinct extents by dierent stimuli (reviewed in Cooper, 1994; Cano and Mahadevan, 1995; Seger and Krebs, 1995). The three MAP kinase subtypes dier in their abilities to phosphorylate transcription factors, such as Ternary Complex Factor (TCF), c-Jun and ATF-2, which are strongly implicated in controlling c-fos and c-jun induction (reviewed in Cooper, 1994; Cano and Correspondence: LC Mahadevan Received 8 May 1997; revised 8 July 1997; accepted 10 July 1997 Oncogene (1997) 15, 2321 ± 2331  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00